The brain extracellular fluid (ECF) has an important role in maintaining the brain microenvironment for mental health and function. It contains proteins and peptide factors that play a crucial role in certain aspects of brain growth, development and differentiation as will as the modification of neural circuits during learning and long-term memory formation. In previous studies we identified the ependymins as prominent glycoproteins which are concentrated in the goldfish and mammalian (including human) brain ECF and cerebrospinal fluid (CSF). They contain 10% carbohydrate as an N-linked glycan. Ependymins have been implicated in certain aspects of synaptic changes associated with memory consolidation and neuronal regeneration. We have devised an affinity chromatographic method for the isolation of ependymins in milligram quantities. Such preparations will be used in structure and properties studies. The composition and sequence of the glycan and peptide fragments, generated by protease digests, of the molecule will be determined. In previous studies we found that the ependymins can rapidly polymerize to form insoluble fibrous aggregates when calcium is removed from solution by the addition of a chelating agent or dialysis. The products, once formed, were found to be extremely insoluble in a variety of solvents including boiling 1% SDS in 6 M urea, acetic acid, and even trifluoroacetic acid. They were, however, soluble in formic acid. We will investigate the factors that control the polymerization of ependymins looking for the participation of other ECF components, post-translational modifications of the molecule, and the role of its glycan fragment. Several antibodies that differentially recognize the polypeptide and carbohydrate epitopes of the molecule have been prepared and will be used in these studies. We will then use immunocytochemical methods at the light and electron microscope level to determine whether the polymerization occurs in vivo. Such studies will help to establish whether ependymin is used in the formation of an extracellular matrix and define its possible role in synaptic and connectivity phenomena. This research may help explain the mechanisms by which the central nervous system maintains its connectivity for mental health and function and how it establishes new synapses during growth, development and memory formation.